LU82623A1 - PROCESS FOR THE PREPARATION OF BIOCOMPATIBLE MATERIALS BY THE SURFACE IMMOBILIZATION OF THE APYRASE, AND ARTICLES MANUFACTURED THEREBY - Google Patents

Description

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Process for the preparation of biocompatible materials by the surface immobilization of the apyrase, and manufactured articles obtained by this process.

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The present invention relates to a process for the preparation of biocompatible polymeric and non-polymeric materials by immobilizing apyrase on their surface, apyrase being an enzyme capable of catalyzing the conversion of adenosine diphosphate (ADP) to adenosine monophosphate ( AMP), then the conversion of the latter into adenosine. The invention also relates to the manufactured articles obtained by said process.

The possibility of imparting biocompatible properties to various types of materials is of enormous practical importance at present. In this respect, there are materials which, because of their good mechanical properties, machining and resistance, and the absence of toxicity, find immediate application in the construction of prostheses of medium or long duration for implantations, or in the construction of elements of auxiliary machines used in extracorporeal circulation, such as renal dialysis machines or artificial hearts and lungs.

For this application, the materials which could be used are found in a very wide range, ranging from cellulosic polymers, aliphatic or aromatic polyamides, polyesters, polycarbonates, polyurethanes and PVC to special metal alloys. Unfortunately, the use of these materials is greatly limited by their generally weak biocompatibility. This is due to the fact that as soon as a foreign material is inserted into the bloodstream, it immediately gives rise to the formation of thrombi due to the initiation of an extremely complicated process.

It is assumed that there is first an adhesion of the platelets of the blood to the surface of the material, with as a consequence the elimination of the ADP and of the serotonin of the platelets, which then causes the agglomeration of the platelets . Platelet agglomeration is itself a fundamental stage ”2 This is due to the fact that it gives rise to the elimination of phospholipids which are essential in the process of blood clotting, causing the conversion of fibrinogen to fibrin.

The role of ADP as an inducer of platelet agglomeration, and therefore as a trigger for the thrombi formation process is widely known. See for example the fundamental work of A. Gaarder and A. Hellem in Nature 192, 531 (1961). It is therefore seen that im-mobilization of an enzyme such as apyrase, capable of converting ADP produced into AMP and adenosine produced by the adhesion of platelets to the surface of the material contacted with the blood, can prevent further aggregation of platelets, thereby blocking the formation of thrombi. In this connection, the authors of the present invention have discovered, and this forms the object of the present invention, that the immobilization of the apyrase on the surface of the thrombogenic materials gives the latter satisfactory biocompatible properties.

These properties are independent of the system used to immobilize the enzyme. This immobilization can be done by adsorption and subsequent crosslinking on the surface of the polymeric materials, or even on metallic surfaces, for example on the surface of the needles used for artery-vein connections in the extracorporeal circulation. In addition, apyrase can be linked cova-

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j slow to the functional groups present on the surface of r! materials whose biocompatibility must be increased. It is indeed possible, if necessary, to activate the material at? beforehand in order to release on its surface the reactive groups which can be used to immobilize the apyrase.

For example, the enzyme can be covalently attached! to amino (or carboxyl) groups of aliphatic or aromatic polyamides having undergone gentle surface hydrolysis.

Likewise, the carboxyl groups of the surface hydrolyzed polyesters can be used.

> The present invention is illustrated by the following descriptive and nonlimiting examples.

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Example 1

"Nylon" rings 6 (length 9 mm, internal diameter 4 mm, external diameter 5 mm) are prepared, taking care during their machining that the edges are chamfered 5 and rounded.

A 0.25 mm deep groove is made along the middle of the outside of the ring around its entire circumference.

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The rings are hydrolyzed in 3.5 N HCl at 37 ° C for 1 hour.

To ensure that the surface hydrolysis of the "Nylon" has been carried out, a colorimetric test is carried out which consists in immersing the rings, washed with water, in a solution containing 0.1% (weight / volume) d trinitrobenzene sulfonic acid in saturated tetraborate.

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After about 1 hour, the formation of an orange color on the surface of the "Nylon" confirms that the hydrolysis has taken place. At this time, the rings are immersed in a 2.5% glutaric aldehyde solution, and kept immersed for 3.5 hours at 4 ° C. The rings are then washed quickly in ice water and immersed in a 0.01 M phosphate buffer solution containing 8 mg / ml of apyrase (specific activity 3.4 IU / mg using ADP as substrate). The reaction is allowed to take place at 4 ° C overnight and overnight. After the reaction is complete, the rings are washed in distilled water, and the enzyme activity is examined. A ring is immersed in 50 ml of 0.1 M TRIS HCl buffer solution of fold 7.4, containing 0.25 mg / ml of ADP and 0.5 mg / ml of CaCl 2.

The mixture is kept stirring at 25 ° C. 1 ml samples are taken every 30 minutes and are analyzed in order to determine the mineral phosphates according to the method of Piske and Subbarrow (C.H. Fiske; Y. Subbarow; J.

Biol. Chem. 66, 375 (1925). An enzyme activity of 4.5 µmoles of mineral phosphates removed in 1 hour is measured on the rings.

J · The "Nylon" rings with the apyrase covalently linked to the superficial amino groups are subjected to an "in vivo" biocomoatibility test by inserting them into the 4 femoral vein of medium-sized dogs.

. The insertion is made, under general anesthesia, and when the ring has been inserted into a vein, it is fixed by means of a silk thread. The wound is sutured and antibiotics are administered to the animals.

A similar untreated "Nylon" ring is inserted for comparison according to the same method. e After an insertion period of 15 days, the rings are extracted and examined.

The ring with apyrase is found to be permeable while the reference ring is completely blocked by thrombi.

A measurement made on the ring with apyrase extracted from the dog shows approximately 4 yumoles of mineral phosphates eliminated in 1 hour.

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Example 2

"Nylon" rings 6 similar to those described in Example 1 are prepared and immersed in a 0.01 M phosphate buffer IS solution of pH 7 containing 8 mg / ml of apyrase.

After 4 hours, double the volume of a 2.5% glutaraldehyde solution in 0.01 M phosphate buffer solution, pH 7, is added.

The mixture is left to act at 4 ° C, losing a day and a night. The rings are then washed and tested for enzymatic activity according to the process described in Example 25 1.

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There is an enzymatic activity of approximately 2 jumps of mineral phosphates eliminated in one hour by a single ring.

The "in vivo" biocompatibility test is carried out by inserting rings with apyrase and reference rings | 30 days in the femoral vein of medium-sized dogs.

The procedure followed for this test is that indicated in Example 1. After an insertion period of 15 days, the rings are extracted. The ring with apyrase is found to be permeable and the enzymatic activity measured is equivalent to about 2 µmoles of mineral phosphates removed in one hour. i The reference "Nylon" ring is however completely blocked. .

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Example 3

Polyethylene terephthalate rings similar to those described in Example 1 are prepared. The rings are hydrolyzed in 1 M NaOH at 50 ° C. for 5 hours. The rings 5 are then washed and immersed in 50 ml of a solution containing 8 mg / ml of apyrase and 10 mg / ml of N-ethyl-N1 (3-dimé-thylaminopropylcarbodiimide) in a phosphate buffer 0.1 < · M at pH 6.8. The reaction is carried out at 4 ° C overnight.

In this way the apyrase is linked to the support by the formation of an amido bond between the--aminolysin groups and the enzyme and the carboxyl groups of the surface hydrolyzed polyester. Examination of the enzymatic activity on the rings, carried out as indicated in Example 1, gives approximately 4 μmoles of mineral phosphates eliminated in 1 hour per ring. The "in vivo" biocompatibility test is carried out by inserting the polyester rings with apyrase and the untreated rings into the femoral vein of experimental dogs. The process described in Example 1 is followed. After 15 days of insertion, the rings are extracted. The reference ring 20 is completely plugged while the ring with apyrase is permeable with only a few sporadic thrombi.

Examination of the enzymatic activity on the ring after insertion "in vivo" gives approximately 3 μmoles of mineral phosphates eliminated in one hour.

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Polyethylene terephthalate rings similar to those described in Example 1 are prepared. The apyrase is crosslinked on these rings according to the process indicated in Example 2. After immobilization, the enzyme activity is found by ring approximately 3 μmoles of mineral phosphates eliminated in one hour. The "in vivo" biocompatibility test on dogs, carried out according to the method described in Example 1, gives. positive results for rings with crosslinked apyrase, which are found to be permeable and almost completely free of thrombi, while the reference rings are completely plugged. The residual enzymatic activity of

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yT rings is about 2.5 yjmoles of mineral phosphates »t i eliminated in one hour.

I Example 5

C 50 steel rings similar to those described in Example 1 are prepared. The apyrase is fixed to a few of them by crosslinking with glutaric aldehyde by the process described in Example 1. The immobilized activity! on each group is measured and happens to be around i "| 1.5 ^ umole of mineral phosphates eliminated in 1 hour. The | "in vivo" biocompatibility tests are carried out on! * 10! experience dogs as previously described.

. After 15 days of insertion, the rings are extracted and examined. While the reference rings appear to be completely plugged, the rings with crosslinked apyrase are found open and with only a few thrombi inside.

The residual activity of the rings treated with apyrase, after insertion, is 0.9 μmol of mineral phosphates eliminated per hour.

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Claims (6)

1. Process for the preparation of biocompatible materials characterized in that it consists in immobilizing on polymer or metal supports an appropriate biological agent capable of eliminating ADP in the blood. 2. Method according to claim 1, characterized by the "‘ fact that the biological agent is the apyrase enzyme. * 3. Method according to claim 1, characterized by the fact that the biological agent is immobilized on the support by * an adsorption and crosslinking process. 4. Method according to claim lf characterized in that the biological agent is immobilized on the support by the formation of a covalent bond between the biological agent 1b and the functional groups existing on the support. 5. Method according to any one of claims 1 to 4, characterized in that the polymeric support is chosen from aliphatic and aromatic polyamides, polyesters and cellulosic polymers and the like, which have undergone gentle hydrolysis. 6. Prosthetic articles, probes, tubes, needles, membranes, various prostheses and artificial organs in general, biocompatible, prepared according to the process described in any of claims 1 to 5. s _